Integrated thermoplastic chip for rapid pcr and hrma

ABSTRACT

The present invention relates to a microfluidic system including a temperature controller and a thermoplastic microfluidic chip that enables rapid PCR in a PCR chamber of the microfluidic chip. Thermal control of the PCR chamber is achieved by applying voltage to heater electrodes patterned directly onto one layer of the microfluidic chip. The temperature controller adjusts the voltage applied to the heater electrodes by changing temperature controller parameters selected to minimize duration of each PCR cycle. Furthermore, simple operation of the microfluidic chip is provided through using an integrated passive capillary valve, requiring minimum operator intervention and eliminating the need for fluidic interfacing, pumping, or metering during chip loading.

This application is a divisional of U.S. patent application Ser. No.15/488,070, filed on Apr. 14, 2017, which claims the benefit of U.S.Provisional Patent Application Ser. No. 62/323,200, filed on Apr. 15,2016, which is incorporated herein by reference in its entirety.

BACKGROUND Field of the Invention

The present invention relates to a thermoplastic microfluidic devicethat enables rapid nucleic acid amplification, such as polymerase chainreaction (PCR), together with high resolution melt analysis (HRMA) ofthe resulting amplified product in a single integrated platform.

Discussion of the Background

Benchtop amplification and PCR platforms commonly employ large resistiveor thermoelectric heating elements for temperature control, resulting inhigh power requirements and slow amplification times due to the largethermal masses involved. Together with high costs associated withbenchtop PCR platforms, these limitations have constrained the wider useof PCR in point-of-care settings. For use as a near-patient diagnostictool the ideal PCR system should support rapid sample-answer times usingindividual clinical samples, while offering simple operation in a smallfootprint. The system should also employ inexpensive and disposable PCRelements to minimize cost and infrastructure requirements, issues ofparticular concern for applications in global health care andresource-limited environments.

Microfluidic technology offers significant potential for overcomingthese constraints and advancing PCR technology for point-of-careapplications. The earliest PCR microsystems consisted of bulk-etchedsilicon reaction chambers with integrated thin film polysilicon heaters,where inherently low thermal mass and high surface area enabled rapidcycle times around 2 min. It is known in the art to employ a range ofmicrofluidic PCR platforms that use integrated thin film heaterspatterned on silicon or glass substrates containing sealed microchannelswithin which amplification occurs. While these microscale platforms havebeen shown to enable rapid PCR cycle times, the fabrication costsassociated with bulk micromachining and sealing of both silicon andglass substrates can be prohibitive for many applications.

In contrast to these materials, thermoplastics offer significantadvantages for the development of low-cost consumable microsystems. Inparticular, thermoplastics may be patterned using exceptionally low-costreplication techniques and a variety of rapid large-area bonding methodsthat are available for sealing the resulting microchannels. While anumber of thermoplastic PCR chips have been reported, the high heatcapacity and low thermal conductivity associated with engineeringthermoplastics have resulted in thermal response times that prohibitrapid PCR. Several techniques based on non-contact heating have beenexplored to address the thermal limitations of thermoplastics, enablingtheir effective use as microfluidic substrates for rapid nucleic acidamplification. For example, Muddu et al. discloses 10 min PCR in athermoplastic substrate using microscale convection to control the localsurface temperature (R. Muddu, Y. a Hassan, and V. M. Ugaz, “Rapid PCRthermocycling using microscale thermal convection,” J. Vis. Exp., no.49, pp. 1-5, January 2011). Giordano et al. demonstrated a cycle time ofless than 4 min by using non-contact infrared heating of a polyimidemicrodevice (B. C. Giordano, J. Ferrance, S. Swedberg, a F. Hühmer, andJ. P. Landers, “Polymerase chain reaction in polymeric microchips: DNAamplification in less than 240 seconds,” Anal. Biochem., vol. 291, no.1, pp. 124-32, April 2001). Similarly, Son et al. employed non-contactplasmonic photothermal heating of a gold thin film to thermocyclepoly-methyl methacrylate microwells, with amplification achieved in 5min. (J. H. Son, B. Cho, S. Hong, S. H. Lee, O. Hoxha, A. J. Haack, andL. P. Lee, “Ultrafast photonic PCR,” Light Sci. Appl., vol. 4, no. 7, p.e280, 2015).

Accordingly, there is a need for a disposable thermoplastic microfluidicchip that enables rapid PCR together with high resolution melt analysis(HRMA) of the resulting PCR product in a single integrated platform.

SUMMARY OF THE INVENTION

The present invention relates to systems and methods for performingrapid amplification of nucleic acid in a reaction chamber, including PCRamplification, of a microfluidic chip. Specifically, in one aspect ofthe invention a microfluidic system comprising a microfluidic chip and atemperature controller is provided. The microfluidic chip comprisesfirst and second thermoplastic layers. The first layer includes at leastone inlet well in fluid communication with a PCR chamber and one or morehydrophilic capillary valves. The second thermoplastic layer has a firstsurface and a second surface. The first surface of the secondthermoplastic layer has one or more heater and sensor electrodespatterned thereon. The heater electrodes control a temperature in thePCR chamber. The second surface of the second thermoplastic layer isbonded to the first thermoplastic layer sealing the PCR chamber. Thetemperature controller controls an input voltage applied to the one ormore heater electrodes based on data provided by the one or more sensorelectrodes indicative of the temperature in the PCR chamber. The inputvoltage is adjusted during each PCR cycle based on controller parametersselected to minimize duration of each PCR cycle.

In yet another aspect of the invention, a method for performing a rapidPCR reaction is provided.

Specifically, the method comprises providing a microfluidic chipcomprising one or more heater and sensor electrodes, at least one inletwell in fluid communication with a PCR chamber, and one or morehydrophilic capillary valves, the heater electrodes controlling atemperature in the PCR chamber. The next step of the method relates torunning a PCR reaction and controlling an input voltage applied to theone or more heater electrodes based on data provided by the one or moresensor electrodes indicative of the temperature in the PCR chamber. Theinput voltage is adjusted during each PCR cycle based on controllerparameters selected to minimize duration of each PCR cycle.

In one embodiment, the controller parameters are selected based on dataproduced by a simulation model prior to a PCR reaction evaluatingthermal performance of the microfluidic chip during a PCR cycle and/oreach stage of a PCR cycle. In yet another embodiment the controller is aPID controller. In a further embodiment, the first and secondthermoplastic layers are fabricated from cyclic olefin polymer or cyclicolefin copolymer. By way of example, the second thermoplastic layer is50 μm thick and the reaction chamber is 200 μm deep. In a further aspectof the invention, the thickness of the second thermoplastic layer andthe depth of the reaction chamber are selected to minimize the durationof a PCR cycle. Those of skill in the art will understand alternativethermoplastic materials having similar properties to those describedherein, which can also be used in the practice of the invention.

In another aspect of the invention, a sample received at the input wellflows towards the PCR chamber by a capillary action, the sample fluidbeing halted at the PCR chamber by the one or more hydrophilic capillaryvalves. By way of example, an expansion angle of the valve is providedat 150°. One or more valve(s) has a width of approximately 50 μm and adepth of approximately 20 μm. In yet another embodiment, the reactionchamber is matched to the one or more heater electrodes. Temperaturesensing in the reaction chamber is performed by applying a small currentto a first sensor electrode while monitoring a voltage drop across asecond sensor electrode. Furthermore, the PCR reaction in the reactionchamber is followed by a high resolution melt analysis (HRMA) performedin the PCR chamber.

In yet another aspect of the invention, a method of manufacturing a chipis provided. Specifically, the method comprises milling channels in analuminum mold; embossing polyetherimide (PEI) mold with aluminum mold;molding a resin layer onto the PEI mold to produce a wafer; exposing thewafer to a solvent and bonding the wafer to a thin film layer to form amicrochannel; spinning a photoresist onto the wafer andphoto-lithographically patterning the photoresist; developing the wafer;and depositing a thin metal layer on top of the wafer to form heater andsensor electrodes.

In one embodiment, the chip is a microfluidic chip that may be used forperforming a PCR reaction and HRMA analysis. By way of example andwithout limitation, the resin layer and the thin film layer arefabricated from cyclic olefin polymer (COP) or cyclic olefin copolymer(COC). In yet another embodiment, the thin film layer is approximately50 μm thick and the microchannel is 200 μm deep.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the subjectmatter of this disclosure. In the drawings, like reference numbersindicate identical or functionally similar elements.

FIG. 1A demonstrates a 3D schematic of a microfluidic chip according tothe present invention.

FIG. 1B demonstrates a close up view of the heater and sensorintersection as shown in FIG. 1A.

FIG. 1C demonstrates a schematic of the hydrophilic capillary valve ofFIG. 1A.

FIG. 2A demonstrates the step of milling channels in aluminum (Al) mold.

FIG. 2B demonstrates the step of embossing polyetherimide (PEI) moldwith alluminium mold.

FIG. 2C demonstrates the step of molding COP resin onto PEI mold.

FIG. 2D demonstrates the step of exposing COP wafer to decalin solventand bonding the COP wafer to the COP thin film.

FIG. 2E demonstrates the step of spinning negative liftoff resist ontoCOP wafer and photolithographically patterning the resist.

FIG. 2F demonstrates the step of developing the COP wafer.

FIG. 2G demonstrates the step of depositing chromium (Cr) and gold (Au)on top of the COP wafer.

FIG. 2H demonstrates final COP wafer with Cr/Au elements.

FIG. 3A is an image of the microfluidic chip according to the presentinvention taken from the bottom of the microfluidic chip.

FIG. 3B is an image of the microfluidic chip according to the presentinvention taken from the top of the microfluidic chip.

FIG. 4 is a plot of meniscus position (mm) versus time (s) for threedifferent capillary loading and flow stop experiments.

FIGS. 5A-5B relate to a parametric evaluation of different chip designsby plotting total rise time for a complete PCR cycle as a function ofchip geometric dimensions.

FIG. 5C demonstrates temperature vs. cycle time plots for the systemusing both open-loop and closed-loop control.

FIG. 6 demonstrates temperature vs. time plots acquired during a PCRcycle, the temperature measured by a thermocouple embedded into reactionchamber (dot line) and sensor electrodes (solid line), respectively.

FIG. 7A demonstrates amplification curves for the PCR chip according tothe present invention, the curves acquired for different concentrationsand cycle times.

FIG. 7B is a plot of crossover point (Cp) vs. initial targetconcentration.

FIG. 8A is a plot of melt curve data collected on the chip according tothe present invention after completing PCR amplification.

FIG. 8B demonstrates a derivative of the spline equation fitting thecurve in FIG. 8A.

FIG. 9 demonstrates three experiments for G6PC amplification product rununder high sensitivity electrophoresis.

FIGS. 10A-10C demonstrates temperature versus resistance plots overthree cycles, respectively (heating shown as white hollow dots, coolingshown as filled black dots).

FIG. 11 is a block diagram of the microfluidic system according to thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has several embodiments and relies on patents,patent applications, and other non-patent references for details knownin the art. Therefore, when a patent, patent application, or othernon-patent reference is cited or repeated herein, it should beunderstood that it is incorporated by reference in its entirety for allpurposes as well as for the proposition that is recited.

The present invention relates to a disposable thermoplastic microfluidicdevice (microfluidic chip) that enables rapid PCR together with highresolution melt analysis (HRMA) of the resulting PCR performed in thesame reaction chamber. In addition to performing rapid PCR and HRMA in alow-cost format, the thermoplastic chips are designed to address severalsystem-level issues which constrain established microfluidic PCRplatforms. In one embodiment, the microfluidic chip is fabricated usinga two-step embossing process compatible with high throughputreplication-based manufacturing processes. By way of example and withoutlimitation, cyclic olefin polymer (COP) or cyclic olefin copolymer (COC)may be used as a thermoplastic with low autofluorescence. Sealing of theCOP microchannels is performed using a COP or COC film layer, therebyminimizing thermal resistance and mass of the PCR chips. Thermal controlis achieved by patterning thin film metal electrodes directly onto thesealing COP film layer for combined temperature sensing and thermalactuation in a low-power format. In yet another embodiment, simpleoperation of the microfluidic chip according to the present invention isprovided through self-loading of reaction volumes using an integratedpassive capillary valve, requiring minimum operator intervention andeliminating the need for fluidic interfacing, pumping, or meteringduring chip loading. In a further embodiment, a fully self-containedsystem using a microcontroller is employed to implement all assay stepsincluding thermocycling using closed-loop control scheme andfluorescence detection for assay readout. The disposable PCR chipaccording to the present invention successfully combines simpleoperation, rapid PCR, precise temperature control for accurate HRMA, andlow cost in a compact format.

FIG. 1A demonstrates a 3D schematic of a microfluidic chip according tothe present invention. The microfluidic chip comprises a first layer 106and a second layer 102. Thin film heater electrodes 104, thin film metalsensor electrodes 122 and 124, and gold-coated spring contact pins 116are patterned directly on one of surfaces of the second layer 102. Thenumber of heater and sensor electrodes is not limited to the number asshown in FIG. 1A as the chip according to the present invention maycomprise any number of heater and sensor electrodes. The first layer 106is a microfluidic layer that comprises a reaction chamber 108, a passivecapillary valve 110, a microchannel 118, an input well 112, and an airvent 114. The number of microfluidic features such as channels,chambers, valves, and wells is not limited the number shown in FIG. 1Aas the chip according to the present invention may comprise any numberof microfluidic features. The second layer 102 is bonded to the firstlayer sealing the microchannel 118 and the reaction chamber 108. In oneembodiment, the first layer 106 and the second layer 102 arethermoplastic layers. By way of example and without limitation, thethermoplastic may be cyclic olefin polymer (COP). In yet anotherembodiment, sealing of the COP microchannel 118 and chamber 108 isperformed using a 50 μm thick COP film layer. By way of example andwithout limitation, the imprinted reaction chamber may be 200 μm deep,while hydrophilic expansion valve regions are 20 μm deep.

Thermal control of a reaction in the reaction chamber 108 is achieved bythe matching thin film heater electrodes 104 and thin film sensorelectrodes 122, 124 patterned between the heater electrodes directlyonto the second layer 102 for combined temperature sensing and thermalactuation. FIG. 1B demonstrates a close up view of the intersection ofthe heater and sensor electrodes patterned on the second thermoplasticlayer 102. Temperature sensing in the reaction chamber 108 may beperformed by applying a small current to the sensor electrode 124 whilemonitoring the voltage drop across the sensor electrode 122. In oneembodiment, the reaction chamber 108 is used for performing a polymerasechain reaction (PCR). By way of example and without limitation, gold maybe used for both the heater and sensor electrodes due to its lineartemperature coefficient of resistance in the PCR temperature range.Other conductive materials having similar linear temperaturecoefficients of resistance as known to those of skill in the art canalternatively be used in the invention as provided herein.

FIG. 1C demonstrates a schematic of the passive capillary valve 110 influid communication with the microchannel 118 and the reaction chamber108. Sample loading is performed by pipetting a sample into the inletwell 112 of the chip. As the sample is deposited into the inlet well112, capillary action pulls the reaction mixture into the microchannel118, resulting in automated filling of the reaction chamber 108. As thesample fills the reaction chamber 108, the liquid reaches a passivehydrophilic expansion valve 110 incorporated into the first chip layer106 at the terminal end of the reaction chamber 108. The capillaryexpansion valve 110 consists of a sudden increase in the channel width,resulting in a Laplace pressure as curvature of the liquid/air interfaceis forced to increase while exiting the valve 110. At a sufficientlyhigh Laplace pressure, the system reaches equilibrium and the fluid flowis halted.

Burst pressure for a capillary expansion valve is determined in part bythe channel dimensions and the expansion angle, defined as the anglebetween the adjacent surfaces at the valve entrance, with largerexpansion angles generally resulting in higher burst pressures. In oneembodiment, to enhance burst pressure in the microfluidic chip accordingto the present invention, the width and depth of the valve region arereduced to 50 μm and 20 μm, respectively, while the expansion angle isset at 150°.

During loading, capillary pumping of a sample results in consistentfilling of the reaction chamber 108, with fluid reaching the expansionvalve 110. Once reaching the valve 110, the capillary filling processhalts and no further motion of the fluid is observed. FIG. 4demonstrates the position of the leading edge of a fluid sample withinthe reaction chamber 108 as a function of the time for three independentfilling experiments. These three experiments are represented by dots,triangles, and squares, respectively. Based on the graphicalrepresentation, it takes approximately 50 s for the sample fluid to fillthe reaction chamber 108. In addition to the graphical representation,images 402-408 demonstrate different stages of the sample fillingprocess. Specifically, in image 402, a sample fluid received at theinput well 112 flows into the microchannel 118 and reaction chamber 108.The sample fluid is gradually fills the reaction chamber 108 in image404 and approaches the valve 110 in image 106. In image 408, theequilibrium is achieved when the sample fluid stops at the capillaryvalve 110. This simple and robust passive loading method serves toreduce operational error while eliminating the need for complex fluidicinterfacing or pumps during sample loading process.

FIGS. 2A-2H demonstrate a process for fabricating the microfluidic chipaccording to the present invention as shown in FIG. 1A. The microfluidicchip may be fabricated from COP or COC due to their high transparency,low autofluorescence, low water absorption, and low gas permeability. Byway of example and without limitation, FIGS. 2C-2H demonstrate theprocess of fabricating the chip from COC. However, any suitable polymer,including COP and COC can be used. The process starts with milling achannel 206 in an aluminum mold 202 as demonstrated in FIG. 2A. Becausechannel features are milled directly into the initial aluminum mold 202,the aluminum surface can be easily polished after mold machining toensure excellent optical quality in the final COP microfluidic chip. Thenext step, as shown in FIG. 2B, is directed to embossing a secondarymold 204 with the aluminum mold 202. Inverse features from the aluminummold 202 are transferred to the secondary mold 204. In one embodiment,the secondary mold 204 is embossed from polyetherimide (PEI) that is ahigh strength and high transition temperature (Tg) thermoplastic. In oneembodiment, to ensure compatibility with the high temperaturesencountered in PCR, a grade of COP with a glass transition temperature(Tg) of approximately 135° C. is selected. The PEI secondary mold 204proved to be very durable over many embossing cycles, and can be easilyreproduced from the aluminum master as PEI mold tolerance degrades.

The next step demonstrated in FIG. 2C is directed to molding COP resin208 onto the secondary PEI mold 204. Specifically, resin pellets areplaced on the PEI secondary mold 204 and heated.

FIG. 2D demonstrates exposing a COP wafer 216 to a solvent and bonding athin film 210 to the COP wafer 216. In one embodiment, the solvent isdecalin. The multilayer substrate is then pressed at in a hot press tocomplete the bonding. The solvent bonding process allows for permanentsealing of the microchannel 206 without any observable deformation ofthe COP sealing film 210.

Thin film gold electrodes for temperature control are fabricated using aliftoff photoresist process. FIG. 2E demonstrates a negative liftoffphotoresist 212 spun onto the COP wafer 216 and photolithographicallypatterned by using an optical mask 214. Alignment marks pre-molded intothe COP microchannel layer 208 were used to position the optical mask214 to precisely align the electrodes 104 under the reaction chamber 108as shown in FIG. 1A. In the next step as shown in FIG. 2F, thephotoresist 212 is developed. After developing the photoresist 212, achromium adhesion layer and a gold layer 222 are sequentially depositedon top of the COP wafer 216 as demonstrated in FIG. 2G. In the finalstep, as shown in FIG. 2H, the photoresist 212 is removed by immersingthe wafer 216 in an acetone bath.

Images of the microfluidic chip fabricated according to the process ofFIGS. 2A-2H are illustrated in FIGS. 3A-3B. FIG. 3A is an image of themicrofluidic chip according to the present invention taken from thebottom of the chip to demonstrate the gold heater and sensor electrodes104, 122, and 124. FIG. 3B is an image of the chip according to thepresent invention taken from the top of the chip to show microfluidicfeatures including the reaction chamber 108, input well 112, vent well114, passive capillary valve 110, and microchannel 118.

Example 1. Microfluidic Chip Fabrication

A master mold was prepared by milling microscale features into a 15 cmsquare plate of 6061 aluminum using a computer numerical controlled3-axis CNC machine (by way of example and without limitation, MDX-650,Roland DGA, Irvine, Calif.). After machining, the aluminum piece waspolished to a mirror finish by chemical-mechanical polishing (by way ofexample and without limitation, METPREP 4, Allied High Tech ProductsInc., Rancho Dominguez, Calif.). Inverse features from the aluminum moldwere transferred to a secondary mold consisting of polyetherimide (byway of example and without limitation, Ultem PEI 1000) by embossing at230° C. and 225 psi for 30 min using a hot press (by way of example andwithout limitation, AutoFour/15, Carver, Inc., Wabash, Ind.).

The microfluidic substrate was constructed of COP (by way of example andwithout limitation, Zeonex 1420R, Zeon Chemicals, Louisville, Ky.).Approximately 15 mL of resin pellets were placed on the PEI secondarymold and heated to 190° C. for 30 min in the hot press. Once heated, thepressure applied to the stack was increased in 50 psi increments every10 min, and held at a final pressure of 250 psi for an additional 10min. The resulting COP plaque had a thickness of approximately 550 μm.The imprinted reaction chamber was 200 μm deep, while hydrophilicexpansion valve regions were 20 μm deep. Input ports were manuallydrilled using a drill press. The microfluidic substrate was sealed by a50 μm thick COP film (by way of example and without limitation, Zeonex1420R) using a solvent bonding technique modified from a previouslydeveloped procedure (T. I. Wallow, A. M. Morales, B. A. Simmons, M. C.Hunter, K. L. Krafcik, L. A. Domeier, S. M., Sickafoose, K. D. Patel,and A. Gardea, “Low-distortion, high-strength bonding of thermoplasticmicrofluidic devices employing case-II diffusion-mediated permeantactivation.,” Lab Chip, vol. 7, no. 12, pp. 1825-1831, 2007). Thechannel side was exposed to 35% decahydronaphthalene in ethanol (w/w)for 7 min, rinsed with 100% ethanol, and blown dry with N2. Themultilayer substrate was then pressed at 200 psi and 50° C. for 15 minin a hot press to complete the bonding. Thin film gold electrodes fortemperature control were fabricated using a liftoff resist process.Negative liftoff photoresist (by way of example and without limitation,NR9-3000PY, Futurrex Inc., Franklin, N.J.) was spun to a thickness of3.7 μm on the exposed surface of the 50 μm COP layer and patterned bycontact photolithography. Alignment marks pre-molded into the COPmicrochannel layer were used to position the mask to precisely align theelectrodes under the reaction chamber. After developing the photoresist(by way of example and without limitation, RD6 developer, Futurrex Inc.,Franklin, N.J.), a 15 nm chromium adhesion layer and 75 nm gold layerwere sequentially deposited by e-beam evaporation (by way of example andwithout limitation, Denton Vacuum Explorer, Moorestown, N.J.), andphotoresist was removed by immersing the wafer in an acetone bath underlight agitation. The metallized COP wafer was cleaned with methanol,isopropanol, and deionized water, and individual chips (six to a wafer)were separated by CNC milling.

The term “ultrafast PCR” commonly refers to a PCR assay providingamplification times below 10 min. For a typical assay requiring 30cycles for complete amplification, this implies a required cycle time onthe order of 20 s. To determine geometric and thermal control parametersrequired for the thermoplastic PCR chip according to FIGS. 3A-3B toreach this target, numerical simulation models are used to evaluate theimpact of chip dimensions on thermal response of the system. Atemperature controller is used to dynamically control input voltage forheating elements (for example, heating electrodes 104 in FIG. 1A) inresponse to thermal sensors readings (for example, sensors 122, 124 inFIG. 1A). Temperature controller parameters are selected to minimize theduration of a PCR cycle. In one embodiment, the temperature controllerparameters are selected based on a simulation model evaluating thermalperformance of a microfluidic chip (for example, the chip of FIGS.3A-3B) during a PCR cycle and/or each step (denature, annealing,extension) of the PCR cycle. Accordingly, the temperature controllerparameters are selected prior to performing a PCR reaction and are fixedduring the PCR reaction. The input voltage for heating elements isdynamically adjusted during the PCR reaction based on the preselectedtemperature controller parameters. The duration of each PCR step(denature, annealing, extension) is set in temperature controllersoftware, with temperature held at a given set point for a fixed time.The temperature controller parameters dictate how quickly thetemperature changes between PCR steps, and helps to maintain the settemperature once reaching the set point.

Realistic values are used for forced convective cooling of the chipsurface and current density limits for the heater electrodes. Rise time,defined as the time required for the response to rise from 10% to 90% ofthe steady state value, is extracted from simulation models for variouschip designs over a range of thicknesses for the COP sealing film 102(FIG. 1A) as well as a range of reaction chamber depths in themicrofluidic substrate 106 (FIG. 1A).

FIGS. 5A-5B result from a simulation model and allow to evaluatedifferent chip designs by plotting the total rise time for a completePCR cycle as a function of different chip parameters. Specifically, inFIG. 5A, the reaction chamber depth was kept constant at 200 μm whilebottom thickness was varied from 50 to 400 μm. In FIG. 5B, the bottomthickness was kept constant at 50 μm while the reaction chamber depthwas varied from 50 to 500 μm. FIG. 5C provides temperature vs. cycletime plots for the microfluidic chip of FIGS. 3A-3B using both open-loop(dashed line) and closed-loop (solid line) control system during a PCRreaction. The cycle time for closed-loop control system is 18.5 seconds,while the cycle time for open-loop control system is 80 seconds.

Based on results as presented in FIGS. 5A-5C, a direct correlationbetween each geometric parameter of the chip and the thermal rise timewas observed, suggesting that smaller sealing layer thickness andchamber depth are desired. In one embodiment, chips were fabricatedusing a sealing layer thickness of 50 μm since the impact of thisparameter on thermal response for lower thickness values is minimal.Sealing films below 50 μm proved challenging due to film deformationduring bonding. In yet another embodiment, for the reaction chamber, adepth of 200 μm was selected as a tradeoff between rapid thermalresponse and high optical path length for sensitive fluorescencemeasurements. In practice, fluorescence intensity was not found to be alimiting factor for the system, indicating that further designs couldfurther enhance thermal response times through the use of shallowerreaction chambers.

The data in FIGS. 5A-5B is presented for open-loop control of a PCRreaction. FIG. 11 relates to closed-loop control of a PCR reaction andillustrates a functional block diagram of a system including amicrofluidic device (chip) 1102 and a main controller 1114. A DNA sampleis input in the microfluidic chip 1102 to undergo a PCR reaction andsubsequent post-PCR analysis. The main controller 1114 includes atemperature controller 1106 and imaging system controller 1114. Thetemperature controller 1106 is configured to provide closed-loop controlfor all assay steps including thermocycling of the PCR chamber 108 (FIG.1A). The temperature controller 1106, which may be a programmed computeror other microprocessor, sends signals to a heating system 1110,including the heater electrodes 104 (FIG. 1A), based on the temperaturedetermined by the temperature sensors 122, 124 (FIG. 1A). In this way,the temperature in the PCR chamber 108 (FIG. 1A) can be maintained at adesired level. In one embodiment, the temperature controller 1106provides instruction on controlling the temperature in the PCR chamberto minimize duration of a PCR cycle. By way of example and withoutlimitation, the temperature controller 1106 may be a PID control system.Other aspects of suitable temperature control system in accordance withthe present invention are disclosed in U.S. Pat. No. 9,061,278,incorporated herein by reference in its entirety.

According to some embodiments of the present invention, the PCR chamber108 (FIG. 1A) may also be cooled by a cooling device 1108, which mayalso be controlled by the temperature controller 1106. In oneembodiment, the cooling device 1108 may be a heat sink or forcedconvection air cooled device, for example. To monitor the PCR processand HRMA analysis that occur in the PCR chamber 108 (FIG. 1A), themicrofluidic system according to the present invention may include animaging system 1104 and an imaging system controller 1116. The imagingsystem 1104, including an image capturing device (by way of example andwithout limitation, CCD camera) and an excitation source (by way ofexample and without limitation, LED source), is in optical communicationwith the PCR chamber 108 (FIG. 1A) to acquire fluorescence images duringa PCR reaction and HRNA analysis. Other aspects of a suitable imagingsystem in accordance with some aspects of the invention are disclosed inU.S. Pat. No. 8,058,054, incorporated herein by reference in itsentirety.

The temperature controller 1106 dictates the input voltage applied tothe heater electrodes based on feedback from temperature sensorelectrodes built into the model. In one embodiment, when the controlsystem is a PID control system, the PID parameters are optimizedmanually, with the maximum input voltage limited to account for thephysical constraints of the actual heating elements and based on thefabricated chip dimensions. FIG. 5C produced by a simulation modeldemonstrates that closed-loop control significantly decreases theduration of a PCR cycle compared to open-loop control.

To confirm the closed-loop numerical model used in FIG. 5C, thermalperformance was characterized in a fabricated microfluidic chipaccording to the present invention (FIG. 1A and FIGS. 3A-3B) containingboth thin film temperature sensors and a thermocouple embedded in thereaction chamber during chip sealing. PID parameters, identical to theparameters used in FIG. 5C, were used for the software-definedclosed-loop control scheme executed by the temperature controller 1106.A comparison of model results with data from both the sensor electrodesand thermocouple is presented in FIG. 6. It is evident that the initialparts of both denature and anneal steps exhibit a dynamic offset betweenthe thin film sensor and the reaction chamber thermocouple measurements.This offset is compensated for in software. Using this approach, a cycletime of 14 s was achieved, slightly faster than a modeled cycle time of19 s when employing identical dwell times at each temperature set point,and well within our targeted time of 20 s per cycle.

In one embodiment, to produce the data as shown in FIGS. 5A-5C, a 3Dmodel (by way of example and without limitation, Version 4.4, COMSOL,Burlington, Mass.) incorporating PID controls and chip design parameterswas used to study thermal performance during each step (denature,anneal, and extension) of the PCR cycle. The PID algorithm used PCRstep-specific parameters to maximize speed and control authority at eachstep. The input voltage, clipped to limit the current density throughthe thin film elements, was determined by using feedback from atemperature sensor electrode built into the model. Dimensions of thebottom COP film thickness and reaction chamber depth were varied overranges that were selected based on material and fabrication processconstraints. Forced air convection was incorporated into the model torepresent high CFM fans blowing on the chip during operation, withconvective heat flux parameters for the denature, anneal, and extensionsteps. In one embodiment, the convective heat flux parameters fordenature, anneal, and extension steps were given by 40, 80, and 40W²K⁻¹, respectively. Rise time was determined using the model at eachthermocycle step for each geometric design variation. The model was alsoused to compare cycle times under open-loop and closed-loop control. Forclosed-loop control, the input voltage was determined using a PIDcontrol algorithm and temperature feedback from the sensor element.Proportional, integral, and derivative controller constants were variedto optimize cycling speed and control authority at each PCR step. In oneembodiment, total cycle times for both open-loop and closed-loop controlincluded hold times at the denture, anneal, and extension steps of 2, 2,and 4 s, respectively.

In yet another embodiment, a microcontroller platform 1114 (FIG. 11) (byway of example and without limitation, Atmel ATmega328P-based platform,ProTrinket 5V, Adafruit, New York, N.Y.) was programmed to implement allassay control functions, including temperature sensing, control overvoltage applied to the heating electrodes (for example PID control), andtriggering of a detection system including CCD camera (by way of exampleand without limitation, DMK41BU02, Imaging Source, Charlotte, N.C.) andLED light source (by way of example and without limitation,2600N-701-14-C2, Innovations in Optics, Woburn, Mass.) for fluorescenceimaging. In one embodiment, the PID control algorithm outputs a pulsewidth modulated signal to adjust average DC power applied to the heaterswith an n-type MOSFET. The integrated closed-loop temperature controller1106 (FIG. 11) and data acquisition system 1104 (FIG. 11) were then usedto implement software-defined PCR and HRMA routines.

In one embodiment, temperature sensing in the reaction (PCR) chamber 108of the microfluidic chip according to FIG. 1A and FIGS. 3A-3B isperformed by applying a small current to the sensor electrode 122 (FIG.1A) while monitoring the voltage drop across the sensor 124 (FIG. 1A).The PCR chamber 108 is matched to a pair of heating electrodes 104, witha single sensor electrode placed between them (FIG. 1B). A low 1 mAcurrent is used to prevent Joule heating of the sensor electrode. In oneembodiment, gold is used for both the heating and sensing electrodes dueto its linear temperature coefficient of resistance in the PCRtemperature range. To maximize temperature resolution, a sensorinterface was used to convert the raw input signal range to the full 5 Vanalog input range of the microcontroller using a level shift amplifiercircuit to yield zero output voltage at 55° C. together with aninstrumentation amplifier circuit with a voltage gain of 250. In afurther embodiment of the present invention, the sensor interfacecircuit and microcontroller connections to the various system componentswere assembled on a custom circuit board with a total footprint below 8cm square, including the microcontroller itself. Electrical connectionsbetween the controller board and thermoplastic chips are made withsurface-mount gold-coated spring contact pins.

Before testing, each device is calibrated to enhance temperatureaccuracy. Chips were first burned in by looping the temperature between23° C. and 95° C. for at least 3 cycles at a ramp rate of ±1° C./s,after which the temperature coefficient of resistance (TCR) for eachchip is determined. The TCR is dependent on gold microstructure, whichcan vary within individual wafers, across multiple wafers in a singlefabrication run, and between batches from different runs. Calibrationwas performed by placing the chips in an oven with a thermocouple andvoltmeter attached to measure resistance and temperature. While in oneembodiment this process was performed for individual chips, calibrationcan be readily automated for large numbers of chips in parallel.

FIGS. 10A-10C represent heater resistance as a function of temperatureduring first, second, and third cycle, respectively. Burn-in of theelectrodes is critical for achieving accurate temperature measurementsand high sensing resolution. As demonstrated in FIG. 10A, prior toburn-in, significant hysteresis was observed for the sensors, with up to3% variation in sensor resistance at 60° C. After three burn-in cycles(FIGS. 10A-10C), hysteretic variations were reduced to less than 2%within the full PCR temperature range. The TCR of each sensor wasdetermined after burn-in for each individual chip. This calibrationprocedure is necessary due to large chip-to-chip variability. Evenwithin a single COP wafer containing six individual microfluidic chips,nominal resistance values were found to vary with relative standarddeviations up to 4%. Burn-in is a standard process step in themanufacture of many temperature sensors such as discrete thermistors,and can be implemented in a highly parallel process for low costfabrication.

During PCR, fluorescence images are collected by the imaging system 1104(FIG. 11) at the end of an extension step during each PCR cycle. Imagedata was communicated over a serial port to a computer and averagefluorescence within the reaction chamber was plotted in real time. Inone embodiment, the threshold cycle (Ct) was calculated independentlyfor each data set as the cycle number at which the fluorescence signalreached a value of 20σ, where σ is the standard deviation of thefluorescence signal over cycles 3-15. After amplification, HRMA analysiswas performed on the amplified nucleic acids. In one embodiment, duringHRMA analysis, the chip temperature was ramped from 60° C. to 85° C. ata rate of 0.07° C./s with fluorescence images collected every 5 s. Aspline curve was fit to the fluorescence data and the derivative of thecurve is taken at each temperature point. The melting temperature (Tm)was determined as the temperature where the derivative is maximum.

A G6PC assay comprising forward and reverse primers together with custommaster mix including DNA-intercalating dye is used to evaluate themicrofluidic chip as demonstrated in FIGS. 3A-3B. The human G6PC gene isassociated with type I glycogen storage disease (von Gierke's disease),a metabolic disorder resulting in the accumulation of glycogen and fatin body tissues and low blood glucose levels.

Example 2—PCR and HRMA in a Microfluidic Chip

A commercial G6PC assay (by way of example and without limitation, G6PCc.79delC Novallele Genotyping Assay, Canon U.S. Life Sciences, Inc.,Rockville, Md.) was used to evaluate the microfluidic chip according tothe present invention. By way of example and without limitation, samplesof human genomic DNA (hgDNA) (id # NA11254) were purchased from CoriellInstitute for Medical Research (Camden, N.J.). The G6PC primer set,mastermix, and hgDNA template were added in equal volumes to eachreaction volume for a final concentration of 20 ng/μL. The hgDNA wasdiluted in Tris-EDTA buffer for the lower concentration reaction.

The software-defined PCR routine implemented by the microcontrollerconsisted of a 30 s hot start at 95° C., followed by 35 cycles of 95° C.for 5 s, 66° C. for 4.5 s, and 72° C. for 4.5 s. A final extension stepat 72° C. for 30 s was performed on cycle 35. The total PCR run time wasapproximately 8.5 min. Separate chips were used to run the assay with10³, 2×10³, and 10⁴ copies of initial template. Identical dilutions at10⁴ copies were run at both 30 s and 14 s cycle times to evaluate theimpact of dwell times on amplification. The resulting amplificationcurves acquired at different initial target concentrations and cycletimes are presented in FIG. 7A.

Extracted from the data of FIG. 7A Cp values (Cp, “crossover point” or“threshold cycle,” refers to the number of cycles it took to detect areal signal from a sample) as a function of initial target concentrationare shown in FIG. 7B. Cp values for each test are calculated by takingthe standard deviation (σ) of the background fluorescence during cycle's3-15 for each data set. This σ was multiplied by a factor of 20 todetermine a threshold value for each independent data set. No differencein Cp was observed for the case of 10⁴ copies at 14 s and 30 s,indicating that the shorter cycle time is sufficient to achieveefficient on-chip amplification for the G6PC assay.

In one embodiment, validation of PCR product was performed by using acapillary gel electrophoresis system (by way of example and withoutlimitation, BioAnalyzer 2100, Agilent, Santa Clara, Calif.) inconjunction with an assay for detecting a single mutation in the humanG6PC gene. To validate the reaction, capillary gel electrophoresis wasperformed on PCR product of the human G6PC gene extracted from threedifferent microfluidic chips. The analysis, as shown in FIG. 9, revealed51 bp fragments for the microfluidic chips, which compares well with atheoretical G6PC amplicon length of 45 bp.

In yet another embodiment, validation of a PCR reaction performed on themicrofluidic chip according to the present invention employed HRMAtechnique. Specifically, HRMA is performed in the PCR chamber 108 of themicrofluidic device of FIGS. 3A-3B. In the HRMA technique, controlleddenaturing of amplicons with high temperature resolution enablessequence-specific DNA melting temperatures to be evaluated. The meltingtemperatures provide additional information about the PCR productwithout the need for an additional instrumentation or use of a post-PCRassay. Using the integrated microfluidic chips according to the presentinvention, HRMA analysis was completed in 6 min. A typical melt curveresulting from on-chip HRMA analysis of the G6PC product immediatelyfollowing PCR amplification is shown in FIG. 8A. Specifically, in FIG.8A, the fluorescence measured during nucleic acid dissociation ispresented as a function of the temperature. The curve in FIG. 8A wasthen fit with a spline to mitigate sensor noise that results in anon-smooth melt peak. HRMA analysis was done by taking the negativederivative of the spline fit equation at each respective data point andplotting the curve. The maximum (−dF/dT) in fluorescence is identifiedas the amplicon melting temperature. The final HRMA plot consisting ofthe derivative of the melt curve of FIG. 8A is presented in FIG. 8B. Theaverage melting temperature determined for all tested devices was75.14±0.45° C., which compares very well against the theoretical melttemperature.

Accordingly, a disposable microfluidic chip with integrated gold thinfilm heating and sensing electrodes is provided. By taking advantage ofnumerical modeling, an optimized chip design can achieve cycle times of14 s, with a complete 35 cycle PCR assay including HRMA is performed ina total of 15 min. Amplification of hgDNA targeting a mutation in theG6PC gene indicative of von Gierke's disease was successfully performedusing the thermoplastic chips, with on-chip HRMA serving to verify amean G6PC melting temperature of 75.14° C.

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various non-limiting embodiments of thepresent invention.

The present invention has several embodiments and relies on patents,patent applications and other references for details known to those ofthe art. Therefore, when a patent, patent application, or otherreference is cited or repeated herein, it should be understood that itis incorporated by reference in its entirety for all purposes as well asfor the proposition that is recited.

As used herein, any descriptions of any type of genetic material canmean any nucleic acid, including DNA and RNA, in the alternative or theadditive to the genetic material originally provided. Thus, geneticmaterial may include a gene, a part of a gene, a group of genes, afragment of many genes, a molecule of DNA or RNA, molecules of DNA orRNA, a fragment of a DNA or RNA molecule, or fragments of many DNA orRNA molecules. Genetic material can refer to anything from a smallfragment of DNA or RNA to the entire genome of an organism.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. Variations of thoseembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description. The inventors expect skilledartisans to employ such variations as appropriate, and the inventorsintend for the invention to be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

1. A microfluidic system comprising: a microfluidic chip comprising: afirst thermoplastic layer including at least one inlet well in fluidcommunication with a PCR chamber and one or more hydrophilic capillaryvalves; a second thermoplastic layer having a first surface and a secondsurface, the first surface in thermal communication with a heatingelement, the heating element controlling a temperature in the PCRchamber; wherein the second surface of the second thermoplastic layer isbonded to the first thermoplastic layer sealing the PCR chamber; atemperature controller controlling an input voltage applied to the oneor more heater electrodes based on data provided by the one or moresensor electrodes indicative of the temperature in the PCR chamber; andwherein the input voltage is adjusted during each PCR cycle based ontemperature controller parameters to minimize a duration of each PCRcycle.
 2. The system of claim 1, wherein the temperature controllerparameters are selected to minimize the duration of each PCR cycle. 3.The system of claim 2, wherein the temperature controller parameters areselected prior to performing a PCR reaction based on data produced by asimulation model evaluating thermal performance of the microfluidic chipduring each stage of a PCR cycle.
 4. The system of claim 1, wherein theheating element comprises one or more heater and sensor electrodespatterned on the first surface of the second thermoplastic layer.
 5. Thesystem of claim 1, further comprising a cooling device to cool the PCRchamber.
 6. The system of claim 1, wherein the temperature controller isa PID controller.
 7. The system of claim 1, wherein the first and secondthermoplastic layers are fabricated from cyclic olefin polymer (COP) orcyclic olefin copolymer (COC).
 8. The system of claim 1, wherein asample received at the input well flows towards the PCR chamber by acapillary action, the sample fluid being halted at the PCR chamber bythe one or more hydrophilic capillary valves.
 9. The system of claim 1,wherein the second thermoplastic layer is 50 μm thick.
 10. The system ofclaim 1, wherein the reaction chamber is 200 μm deep.
 11. The system ofclaim 1, wherein the reaction chamber is matched to the one or moreheater electrodes.
 12. The system of claim 4, wherein temperaturesensing in the reaction chamber is performed by applying a small currentto a first sensor electrode while monitoring a voltage drop across asecond sensor electrode.
 13. The system of claim 1, wherein an expansionangle of the valve is provided at 150°.
 14. The system of claim 1, oneor more valve(s) has a width of approximately 50 μm and a depth ofapproximately 20 μm.
 15. The system of claim 1, wherein the thickness ofthe second thermoplastic layer and the depth of the reaction chamber areselected to minimize the duration of a PCR cycle.
 16. The system ofclaim 1, wherein the PCR reaction in the reaction chamber is followed bya high resolution melt analysis (HRIVIA) performed in the PCR chamber.17. A method of manufacturing a chip, the method comprising: millingchannels in an aluminum mold; embossing polyetherimide (PEI) mold withaluminum mold; molding a resin layer onto the PEI mold to produce awafer; exposing the wafer to a solvent and bonding the wafer to a thinfilm layer to form a microchannel; spinning a photoresist onto the waferand photo-lithographically patterning the photoresist; developing thewafer; and depositing a thin metal layer on top of the wafer to formheater and sensor electrodes.
 18. The method of claim 17, wherein thechip is microfluidic chip.
 19. The method of claim 17, wherein the resinlayer and the thin film layer are fabricated from COC or COP.
 20. Themethod of claim 17, wherein the thin film layer is 50 μm thick.
 21. Themethod of claim 17, wherein the microchannel is 200 μm deep.